The present invention is concerned generally with cellular compositions and methods for improving cardiac function in a living subject after the occurrence of a myocardial infarction; And is focused in particular upon the preparation and therapeutic use of novel genetically altered mammalian embryonic stem cells and their direct descendent progeny as cells for in-vivo transplantation into the infarcted areas of the myocardium in a living host subject.
Myocardial infarction (MI) is a life-threatening event and may cause cardiac sudden death or heart failure. Despite considerable advances in the diagnosis and treatment of heart disease, cardiac dysfunction after MI is still the major cardiovascular disorder that is increasing in incidence, prevalence, and overall mortality (Eriksson et al., 1995). After acute myocardial infarction, the damaged cardiomyocytes are gradually replaced by fibroid nonfunctional tissue. Ventricular remodeling results in wall thinning and loss of regional contractile function. The ventricular dysfunction is primarily due to a massive loss of cardiomyocytes. It is widely accepted that adult cardiomyocytes have little regenerative capability. Therefore, the loss of cardiac myocytes after MI is irreversible. Each year more than half million Americans die of heart failure. The relative shortage of donor hearts forces researchers and clinicians to establish new approaches for treatment of cardiac dysfunction in MI and heart failure patients.
Cell transplantation has emerged as a potential novel approach for regeneration of damaged myocardium in recent several years. Transplanted cardiomyocytes have been shown to survive, proliferate, and connect with the host myocardium (Soonpaa et al., 1994). Engrafted cells may regenerate new cardiomyocytes to replace infarcted myocardium or serve as a source for therapeutic gene transfer to infarct areas (Leor et al., 1997). Li and his coworkers (Li et al., 1996) demonstrated that transplanted fetal cardiomyocytes could form new cardiac tissue within the myocardial scar induced by cryoinjury and significantly improve heart function (Li et al. 1997). Bishop et al. (Bishop et al., 1990) reported that embryonic myocardium of rats could be implanted or cultured. They suggested that the engrafted embryonic cardiomyocytes proliferated and differentiated in host myocardium. In a recent review, Hescheler et al. (Hescheler et al., 1997) pointed out that pluripotent ES cells cultivated within embryonic bodies reproduce highly specialized phenotypes of cardiac tissue. Most of the biological and pharmacological properties of cardiac-specific ion currents were expressed in cardiomyocytes developed in vitro from pluripotent ES cells. Electrophysiological characteristics of these cells developed from ES cells were similar to those previously described in adult cardiomyocytes or neonatal mammalian heart cells (Kilborn et al., 1990; Hescheler et al., 1997).
Transplantation of xenogeneic, allogeneic, and autologous cardiomyocytes, skeletal muscle cells, and smooth muscle cells in normal and injured myocardium has been reported in different species. Several studies have demonstrated the feasibility of engrafting exogenous cells into host myocardium, including fetal cardiomyocytes (Soonpaa et al., 1994), cardiomyocytes derived from artial tumor (AT1) (Koh et al., 1993), satellite cells (Chiu et al., 1995), or bone marrow cells (Tomita et al., 1999). These engrafted cells have been histologically identified in normal myocardium up to 4 months after transplantation (Koh et al., 1993). Cells transplanted close to native cardiomyocytes could form intercalated disks. Gap junctions have been found between the engrafted fetal cardiomyocytes and the host myocardium (Soonpaa et al., 1994), thereby raising the possibility of an electrical-contraction coupling between transplanted cells and the host tissue. Recently, cell transplantation has been extended into chemically damaged myocardium in rats with coronary artery occlusion (Scorsin et al., 1996; 2000), or in cryoinjured rats (Li et al., 1996) and dogs (Chiu et al., 1995). More recently, Li and his coworkers (Li et al., 2000) showed that autologous porcine heart cell transplantation improved regional perfusion and global ventricular function after a myocardial infarction. Angiogenesis has been found after cardiomyocyte transplantation, which increases the survival of the donor cells in infarcted myocardium. Tissue engineering is a potential therapy for end-stage organ disease and tissue loss (Kim and Vacanti, 1999). Therefore, ES cell implantation is possible a novel approach for therapy of cardiac dysfunction in myocardial infarct hearts.
Over the last two decades, the morbidity and mortality of heart failure has markedly increased (Tavazzi, 1998). Therefore, finding an effective therapeutic method is one of the greatest challenges in public health for this century. Although there are several alternative ways for treatment of heart failure, such as coronary artery bypass grafting and whole-heart transplantation, myocardial fibrosis and organ shortage, along with strict eligibility criteria, mandate the search for new approaches to treat the disease. Cell transplantation has emerged to be able to increase the number of contractile myocytes in damaged hearts. Cardiomyocytes derived from embryonic stem (ES) cells may be a viable source for donor cardiomyocytes (Robbins et al., 1992). ES cells are pluripotent cells derived from the early embryo and retain the ability to differentiate into all cell types in vitro including cardiac myocytes (Maltsev et al., 1993; Metzger et al., 1994; Lavranos et al., 1995; Rathjen et al., 1998). Cardiac myocytes derived from cultured ES cells exhibit cell morphology, sarcomere formation, and cell-cell junctions similar to those observed in cardiomyocytes developing in vivo (Klug et al., 1996; Westfall et al., 1997).
In addition, by using porcine as the host, Van Meter and his colleges (Van Meter et al., 1995) showed that either transplanted human atrial cardiomyocytes or fetal human ventricular cardiomyocytes can induce the growth of new blood vessels in the graft area and the host ventricle. The increase in microcirculation could provide the grafted cells with blood supplies and remove cellular debris after cardiac injury.
After intramyocardial transplantation, these cells may communicate with their surrounding tissue, signaling the formation of blood vessels to nourish them. These transplanted cells also can differentiate to new functional cardiomyocytes in the infarct area. Therefore, it is in theory perhaps possible that such engrafted cells might restore cardiac function. One more advantage of ES cell transplantation is that less immunorejection reaction may occur because of lack of membrane surface antigens in ES cells. Thus, this unique approach might provide for an effective in-vivo therapy of myocardial infarction and heart failure. If such an unexpected approach were developed and brought into functional existence, the public health benefits of this therapeutic approach would be potentially great, because millions of patients in the world die of MI and heart failure every year.
The present invention has multiple aspects. A first aspect provides a genetically altered embryonic stem cell suitable for on-demand implantation in-vivo into a living host subject, said genetically altered embryonic stem cell comprising:
a primordial embryonic stem cell of mammalian origin which
(i) remains uncommitted and undifferentiated while passaged in-vitro as a self-renewing cell,
(ii) is implantable in-vivo at a chosen anatomic site as an uncommitted cell,
(iii) integrates in-situ after implantation into the body of the living host subject at a local anatomic site, and
(iv) differentiates in-situ after integration into a recognised type of differentiated cell of embryonic cell origin; and
at least one extra nucleotide segment comprising a vector and not less than one DNA sequence encoding a specific product which is to be expressed subsequently by said embryonic stem cell in-situ.
As second aspect of the invention provides the living progeny of a genetically altered embryonic stem cell suitable for on-demand implantation in-vivo into a living host subject, said living progeny comprising:
multipotent descendant cells of mammalian embryonic stem cells origin which
(i) remain undifferentiated while passaged in-vitro as mitotic cells,
(ii) are implantable in-vivo at a chosen anatomic site as undifferentiated cells,
(iii) integrate in-situ after implantation into the body of the living host subject at a local anatomic site, and
(iv) differentiate in-situ after integration into a recognised type of differentiated cell of embryonic cell origin; and
altered mammalian genomic DNA comprising a vector and not less than one DNA sequence encoding a specific product which is to be expressed subsequently by said progeny cells in-situ.